Key words
short-chain fatty acids - oxidative stress - inflammation - diabetic nephropathy
Introduction
In recent years, gut microbiota have emerged as key players in the heightened risks
of a systemic immuno-inflammatory response and in kidney failure progression, which
has been coined the “Gut–kidney axis” [1]. One class of molecules that acts as a link between the microbiota and the inflammatory
response are short-chain fatty acids (SCFAs), the main metabolic products by the bacterial
fermentation of macro-fibrous material that escapes digestion in the upper gastrointestinal
tract and enters the colon [2]
[3]
. 90~95% SCFAs in the colon are made up of acetate, propionate, and butyrate, with
intraluminal concentrations of about 60% acetate, 25% propionate, and 15% butyrate
[4]
. Locally, SCFAs are energy sources for colonocytes and have been recognized as potential
mediators involved in the effects of gut microbiota on intestinal immune function
[5]
. However, SCFAs can reach the bloodstream and are involved in inflammatory and immune
responses by acting on their specific receptors GPR43, and increasing studies are
shedding light on the roles of GPR43 in SCFAs-associated inflammatory diseases such
as inflammatory bowel disease (IBD), asthma, arthritis and other inflammatory diseases
[
[6]
[7]
.
Systemic and local low-grade inflammation, generation of reactive oxygen species (ROS)
and release of pro-inflammatory cytokines induced by the metabolism of hyperglycemia
and dyslipidemia are implicated in the development and progression of type 2 diabetes
(T2D) and diabetes nephropathy (DN) [8]
[9]
. Recent, 3 studies [10]
[11]
[12]
have attempted to therapeutically use SCFAs or their derivatives in systemic immunologic
and inflammatory responses of kidney injury in both animal and cell models. Accordingly,
therapies have been proposed to restore SCFAs production or to provide exogenous SCFAs
in an attempt to reduce inflammation. However, it is currently unknown whether SCFAs
or GPR43 agonist inhibit oxidative stress and inflammatory response in glomerular
mesangialn cells (GMCs) induced by high glucose and LPS. Therefore, we observed the
effects of the SCFAs or GPR43 agonist treatment on the protein expression of membrane
receptor GPR43 and adhesion molecule ICAM-1, and detected the release of pro-inflammatory
cytokine MCP-1 and IL-1β from GMCs stimulated by high glucose and LPS, and then analysised
of the generation of oxidative stress relevant molecules (ROS, MDA, and antioxidant
enzyme SOD), finally, discussed implications for DN pathogenesis.
Materials and Methods
Renal mesangial cell culture and stimulation
Mouse glomerular mesangialn cells (SV-40 MES 13, obtained from China Center for Type
Culture Collection) were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Glbco)
containing 5.6 mmol/L glucose and 10% fetal bovine serum (FBS, Glbco) at 37°C and
5% CO2.
Initially, to determine proper concentrations of each SCFAs, GPR43 agonist and LPS,
glomerular mesangialn cells were randomly divided and the following treatments were
applied: (1) Lipopolysaccharide (LPS; Sigma-Aldrich) at 0.5, 1, 5, and 10 μg/ml concentrations;
(2) Sodum acetate (Ac; Sigma-Aldrich) at 0.1, 1, 12.5, 25, and 50 mmol/L (mM) concentrations;
(3) Sodium propionate (Pr; Sigma-Aldrich) at 0.1, 1, 12.5, 25, and 50 mM concentrations;
(4) Sodium butyrate (But; Sigma-Aldrich) at 0.1, 1, 5, 10, and 20 mM concentrations;
(5) A phenylacetamide compound (Merckmillipore) acts as an allosteric agonist of GPR43
at 0.1, 1, 10, and 20 μmol/L (μM) concentrations. All of the pH changes in media by
high concentration of SCFAs were adjusted to PH 7.35 by NaHCO3.
Cell viability assay
GMCs were seeded in 96-well plates at concentrations of 1×105 cells/ml to 50% confluence in DMEM complete growth medium, followed by treatments
respectively supplemented with different concentrations of LPS, each SCFAs and GPR43
agonist as described above or mixed LPS and each SCFAs or GPR43 agonist for 24 h in
full media. Cell viability was measured by quantitative colorimetric assay with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT) in 96-well plates. Absorbance at 570 nm was measured for the experimental
groups using a microplate reader. MTT experiments were performed in 5 biological replicates.
DMEM low glucose complete growth medium was used as a normal control (NC group). Cell
viability was expressed as the ratio of the signal obtained from treated cultures
and control cultures. According to MTT results and literatures review, 1 μg/ml LPS,
25 mM high glucose (HG), 25 mM Ac, 12.5 mM Pr, 5 mM But and 1 μM GPR43 agonist were
used in the entire study.
Detection of intracellular ROS level
GMCs (1×104 per well) were seeded in 24-well plates and induced by 25 mmol/L high glucose and
1 μg/mL LPS, the specified concentration of SCFAs or GPR43 agonist was added to the
wells and incubated for 24 h respectively. Intracellular production of ROS was measured
using the ROS assay kit (Beyotime Institute of Biotechnology, China) with oxidation
of 2’,7’-dichlorofluorescin diacetate (DCFH-DA) to fluorescent 2’,7’-dichlorofluorescin
(DCF). The fluorescence images were taken with a fluorescence microscope (Leica, Germany)
and measured in a plate reader with excitation at 488 nm and emission at 525 nm according
to the manufacturer’s instructions. The values were expressed as the mean absorbance
normalized to the percentage of the normal control.
Detection of malondialdehyde (MDA) and superoxide dismutase (SOD)
After incubation with different compounds for 24 h as described above, GMCs were harvested
with 0.25% trypsin, and washed twice with PBS. Then, the contents of MDA and total
SOD were determined using the corresponding detection kits (Lipid Peroxidation MDA
Assay Kit; Total Superoxide Dismutase Assay Kit with WST-8; Beyotime Institute of
Biotechnology, China) according to the manufacturer’s instructions.
Enzyme-linked immunosorbent assay (ELISA)
MCP-1 and IL-1β protein level in the culture supernatant was determined using commercially
available ELISA MCP-1 and IL-1β kits (NeoBioscience, China) according to the manufacturer’s
protocols. MCP-1 and IL-1β protein levels were determined by comparing the samples
to the standard curve generated by the kit.
Protein extraction and western blotting
Total proteins were isolated from GMCs using a total protein extraction kit (Kaiji,
Shanghai, China). The protein concentrations in the cell lysates were determined with
a Bicinchoninic Acid Protein Assay Kit (Pierce Biotechnology, Rockford, USA). Proteins
were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)
and transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore). Immunoblotting
was performed using anti-GPR43 antibody (rabbit; dilution 1:800, Santa Cruz Biotechnology)
and anti-β-actin antibody (mouse; dilution 1:2000, Beyotime, China).
Immunofluorescence
GMCs were grown on coverslips in 6-well plates. After overnight adherence, cells were
incubated with different compounds for 24 h as described above and then were fixed
in 4% paraformaldehyde and then blocked with 5% rabbit serum. The cells were incubated
overnight with the anti-ICAM-1 primary antibodies (goat; dilution 1:100; Santa Cruz
Biotechnology) and incubated for 60 min with secondary antibody conjugated to the
fluorescein isothiocyanate fluorescent dye (dilution 1:200; Boster, China). Images
were taken with a fluorescence microscope (Leica, Germany) and the values of semiquantitative
analysis for average intensity of ICAM-1 were assessed by Image-Pro Plus 6.0 software.
Statistical analysis
Each experiment was repeated at least 3 times using different batches of cells. The
data are expressed as mean±standard deviation (SD). Differences were statistically
analyzed using one-way analysis of variance (ANOVA), followed by the Least Significant
Difference post hoc test for multiple comparisons. A probability value of p<0.05 was considered significant.
Result
The pharmacological concentrations of SCFAs or GPR43 agonist inhibied GMCs proliferation
following LPS treatment
The MTT results showed that 1 μg/ml LPS could induce proliferation of GMCs compared
with NC group (p<0.05), while the higher concentration (5, 10 μg/ml) of LPS group did not promote
the cells proliferation compared with 1 μg/ml LPS group (p<0.05), indicating a severe inflammatory state would lead to the inhibition of cell
growth and stimulation of apoptosis ([Fig. 1a]).
Fig. 1 The effects of SCFAs or GPR43 agonist on GMCs proliferation following LPS treatment
were analyzed by MTT assay. a GMCs were incubated with indicated concentrations (0.5, 1, 5, 10 μg/ml) of LPS for
24 h. b and c GMCs were incubated with different concentration range of acetate (Ac), propionate
(Pr), and butyrate (But) or GPR43 agonist for 24 h. d GMCs were treated with 1 μg/ml LPS alone, or were intervened by specific concentration
of these SCFAs or GPR43 agonist following 1 μg/mL LPS treatment. Data were normalized
with respect to normal control and are expressed as mean±SD (n=5), * P<0.05 compared with the NC group; #
P<0.05 compared with the 1 μg/ml LPS group.
The effect of different concentrations of these SCFAs or GPR43 agonist on GMCs’ viability
was also assessed respectively by MTT assay. We observed that 0.1–50 mM concentration
range of Ac (p<0.05), 0.1–25 mM Pr (p<0.05), 0.1–5 mM But (p<0.05) or 0.1–1 μM GPR43 agonist (p<0.05) promoted the cells proliferation in a dose-dependent manner; however, higher
concentrations of SCFAs (≧10 mM But) or GPR43 agonist (≧10 μM) may inhibit cell viability
(p<0.05) ([Fig. 1b, c]). Considering the cytotoxicity detected by MTT and the pharmacological concentrations
of these SCFAs or GPR43 agonist reported in the literature, we chose the 25 mM Ac,
12.5 mM Pr, 5 mM But and 1 μM GPR43 agonist as the intervention reagent. Thereafter,
the results showed that the intervention effects of these SCFAs or GPR43 agonist on
cell proliferation following 1 μg/ml LPS treatment was significantly lower than that
of LPS stimulation solitar group (p<0.05) ([Fig. 1d]).
The pharmacological concentrations of SCFAs or GPR43 agonist diminished high glucose
and LPS induced MCP-1 and IL-1β release from GMCs
As depicted in [Fig. 2a], after 24 h of stimulation, pro-inflammatory cytokines MCP-1 release from GMCs was
obviously increased in the 25 mM glucose and 1 μg/ml LPS synergy treatment group (HG+LPS
group) compared with exposure to normal-glucose (5.6 mM, NC group) (p<0.05), this
result suggested that synergy between high glucose and LPS could induce inflammatory
response in vitro. However, 25 mM Ac, 5 mM But or 1 μM GPR43 agonist reversed high
glucose and LPS induced MCP-1 release (p<0.05). In accordance with MCP-1, similar profiles of IL-1β released from GMCs were
found, incubation of 25 mM Ac, 5 mM But or 1 μM GPR43 agonist with GMCs cultured in
high glucose and LPS condition resulted in a significant decrease of IL-1β protein
concentration level (p<0.05) ([Fig. 2b]).
Fig. 2 The effects of SCFAs or GPR43 agonist on MCP-1 and IL-1β release from GMCs following
high glucose and LPS stimulation were detected by ELISA. Incubation of specific concentration
of these SCFAs or GPR43 agonist for 24 h with GMCs cultured in 25 mM glucose and 1 μg/ml
LPS (HG+LPS) condition, MCP-1a and IL-1β b protein level in the cell culture supernatant were determined by ELISA-based quantification.
Data are expressed as mean±SD (n=5), * P<0.05 compared with the NC group; #
P<0.05 compared with the HG+LPS group.
SCFAs or GPR43 agonist increased the protein expression of GPR43, but inhibited ICAM-1
in GMCs induced by high glucose and LPS
Compared with NC group, the protein expression of GPR43 in lysates of GMCs was inhibited
by high glucose and LPS stimulation for 24 h (p<0.05), but downregulation of GPR43 was reversed by 25 mM Ac, 12.5 mM Pr, 5 mM But,
or 1 μM GPR43 agonist (p<0.05) ([Fig. 3a]). Moreover, high glucose and LPS stimulation for 24 h markedly increased the ICAM-1
protein level in cytoplasm compared with NC group by immunofluorescence (p<0.05); this trend was reversed by 25 mM Ac, 12.5 mM Pr, 5 mM But, or 1 μM GPR43 agonist
(p<0.05), especially 25 mM Ac and 5 mM But ([Fig. 3b]).
Fig. 3 The effects of SCFAs or GPR43 agonist on the expression of GPR43 and ICAM-1 in GMCs
following high glucose and LPS stimulation. a Incubation of specific concentration of these SCFAs or GPR43 agonist for 24 h with
GMCs cultured in high glucose and LPS condition, the protein expression of GPR43 in
cell lysates were detected by western blotting. Data were normalized with respect
to GAPDH and the gray graphs confirmed these trends. b The expressions of ICAM-1 were detected as green fluorescence in the cytoplasm of
GMCs by immunofluorescence and fluorescence microscope (200×). The values of semiquantitative
analysis for average intensity of ICAM-1 were assessed and the gray graphs confirmed
these trends. Data are expressed as mean±SD (n=3), *P<0.05 compared with the NC group; # P<0.05 compared with the HG+LPS group.
SCFAs or GPR43 agonist inhibited the intracellular production of ROS and MDA, and
reversed decrease of SOD in high glucose and LPS treated GMCs
The fluorescence images for ROS generation and quantitative assay showed that intracellular
ROS ([Fig. 4a]) and MDA ([Fig. 4b]) in the HG+LPS group were obviously higher than in the NC group (p<0.05), while the levels of SOD ([Fig. 4c]) were significantly decreased (p<0.05), indicating that high glucose and LPS may induce excessive oxidative stress
in GMCs. However, the pharmacological concentrations of these SCFAs or GPR43 agonist
inhibited the generation of ROS and MDA and reversed decrease of SOD induced by high
glucose and LPS (p<0.05). In addition, the ratio of SOD/MDA ([Fig. 4d]), which reflect the overall anti-oxidative stress effect of these SCFAs or GPR43
agonist, were calculated to confirme these trends (p<0.05).
Fig. 4 The effects of SCFAs or GPR43 agonist on the intracellular production of ROS, MDA
and SOD in high glucose and LPS treated GMCs. The fluorescence images (100×) and quantitative
assay for ROS a, the contents of MDA b, and SOD c were determined, respectively by detection kits after incubation for 24 h with specific
concentration of these SCFAs or GPR43 agonist. The ratio of SOD/MDA d were calculated to confirme these trends. Data are expressed as mean±SD (n=5), *P<0.05 compared with the NC group; # P<0.05 compared with the HG+LPS group.
Discussion
Taking into account the fact that SCFAs are important energetic substrates for epithelial
cells, the confirmation that they are regulators of their proliferation have already
been expected [5]
[13]]
; but the effect of SCFAs on the proliferation of GMCs is still unknown. It must be
pointed out that the physiological concentrations of SCFAs in colon look quite different
from peripheral circulation, peripheral concentrations of SCFAs in particular propionate
and butyrate are lower than 1 mM because of SCFAs metabolism in colonocytes and liver
[4]
. Most of the anti-inflammatory mechanistic studies of these SCFAs that are discussed
at present have been performed with mM concentrations in vitro, gut or kidney [[6]
[7]
[14]]
. In this study, we initially determined the viability and cell toxicity effects of
SCFAs at physiological and pharmacological concentrations and molar ratios [4]
[15]
. Our results showed that the pharmacological concentration range of SCFAs (0.1–50 mM
Ac, 0.1–12.5 mM Pr, or 0.1–5 mM But), although may be higher than physiological concentrations
in plasm, improved the growth of GMCs, which likely due to the provision of energy
source by SCFAs [5]
. However, the higher concentrations of SCFAs (≧10 mM But) may inhibit GMCs viability,
which may be attributed to high concentration related cytotoxicity and apoptosis.
Previous research has also shown that oral administration of all major SCFAs, when
chronically increased higher than physiological levels in vivo, induce tissue inflammation
in ureteral tissues, leading to kidney hydronephrosis in mice [16]
. Taken together, these evidences indicates that SCFAs may have a dual role in GMCs
proliferation depending on the stimulus concentration, but the mechanism is still
needed to be further studied. Therefore, an exploration of the larger concentration
range of SCFAs in various kidney diseases, such as DN, the leading cause of ESRD,
is necessary. This knowledge may resolve some of these controversies by allowing us
to identify the pharmacological concentration and genuine functions of SCFAs in kidney
tissue, be it pro- or anti-inflammatory.
GMCs proliferation and hypertrophy, ECM accumulation, as well as consequent renal
fibrosis induced by high glucose, AGEs, or LPS have been recognized as major pathogenic
events in the progression of renal failure in DN [17]]
[18]
. In this research, the MTT results showed that LPS (1 μg/ml) could induce proliferation
of GMCs, however, the pharmacological concentrations of SCFAs (25 mM Ac, 12.5 mM Pr, or 5 mM But) or GPR43 agonist (1 μM) intervention
inhibited GMCs proliferation following LPS treatment. There is a contrary to the trend
of these SCFAs or GPR43 agonist at the choosed concentration and LPS (1 μg/ml). Interestingly,
SCFAs, mainly butyrate, present different effects on the growth of normal and tumoral
colonocytes. Butyrate inhibits the growth of cancerous colonic cells, but not of normal
colonocytes and, depending on the concentration, it actually increases the proliferation
of normal colonocytes [13]
. The mechanism proposed for this difference is not clear, may be involved in the
cross-talk of related signaling pathways, such as GPCRs and the inhibition of histone
acetylation (HDACs) [13]
[19]. Similar concentrations of SCFAs (25 mM acetate, 12 mM propionate, or 3.2 mM butyrate)
were used in bone marrow DCs (BM-DCs) stimulated with LPS (20 ng/ml), MM55. k kidney
epithelial cells stimulated with inflammatory cocktail (LPS, 10 μg/ml; zymosan, 10 μg/ml;
IL-6, 50 ng/ml; IL-1β, 50 ng/ml, and TNF-α, 100 ng/ml), and HK-2 human kidney epithelial
cells after hypoxia, these findings indicated that SCFAs treatment diminished hypoxia
and inflammation in these cells [10]
. Therefore, the pharmacological concentrations of these SCFAs or GPR43 agonist as
mentioned above were used in the entire study [7]
[10]
[14].
SCFAs are well known for their anti-inflammatory functions by modulating immune cell
adhesion molecule, chemotaxis as well as cytokine release, finally resulting in inhibition
of leukocyte migration to inflammatory sites. Previous research reported that SCFAs
were able to reduce immune cell infiltration to adipose tissue by preventing inflammatory
cell adhesion and chemotaxis [20]
. Our results demonstrated that incubation of SCFAs, especially acetate and butyrate,
or GPR43 agonist with GMCs cultured in high glucose and LPS condition for 24 h resulted
in a significant decrease of MCP-1, IL-1β, and ICAM-1, suggesting that these SCFAs
or GPR43 agonist treatment, diminished inflammation in kidney tissues and cells.
Oxidative stress and inflammation are inseparably linked as each causes and intensifies
the other, which could cause glomerulosclerosis, tubular atrophy, and fibrosis [21]. Our previous research showed high glucose and LPS prime the NLRP3 inflammasome
and NF-κB inflammatory signaling in GMCs via ROS/TXNIP pathway [22]
[23]. Up to present, several studies have looked into the effect of stress and inflammatory
signaling pathways as effector mechanisms of SCFAs, the anti-inflammatory activity
of SCFAs or GPR43 agonist were shown to inhibit the production of ROS and oxidative
stress in this and other studies. Previous research showsed that activation of GPR43
decreased inflammatory markers, which has been associated with diet-induced obesity
and T2D [24]
[25]. Our data shown that the pharmacological concentration of SCFAs or GPR43 agonist
reversed the high glucose and LPS – induced inhibition of GRP43 protein expression,
further more, these SCFAs or GPR43 agonist also inhibited the intracellular production
of ROS and MDA, and reversed decrease of SOD in GMCs induced by high glucose and LPS.
Similar results are demonstrated that the gene expression levels of GPR43 were increased
after acetate treatment in kidney tissue induced by ischemia-reperfusion injury, and
the these SCFAs treatment inhibited ROS production in HK-2 human kidney epithelial
cells after hypoxia [10]
. By contrast, acetate was shown to promote the release of ROS when added on mouse
neutrophils by activating GPR43 [26]
[27]. As ROS and oxidative stress have been reported be involved in a wide array of inflammatory
conditions, including DN; and GPR43 signaling are positive regulators of high glucose
and (or) LPS-induced inflammatory signaling pathways, including NF-κB and MAPK, both
of which are associated with inflammatory response and kidney injury [10]
[11]
[28] ; these results suggested that the activation of GPR43 may be related to the molecular
mechanisms through which SCFAs exert their anti-inflammatory effects in kidney tissue.
However, many questions regarding the function of these SCFAs and GPR43 receptor remain
unanswered and controversial on account of lack of specific GPCRs blockers, therefore,
potent and selective tools will be required for future studies on SCFAs and their
receptors [29]
.
In conclusion, the present study found that SCFAs, especially acetate and butyrate,
and GPR43 agonist reversed the high glucose and LPS induced mesangial cells proliferation,
ROS and MDA generation, and inflammatory cytokine MCP-1, IL-1β and ICAM-1 release.
These combined results support the hypothesis that SCFAs inhibit oxidative stress
and inflammation in GMCs induced by high glucose and LPS, suggesting that SCFAs or
GPR43 signaling pathway may act as potential therapeutic targets for DN. However,
our studies by no means rule out other potential mechanisms by which SCFAs may take
part in inflammatory response, such as inhibition of HDAC, in view of the fact that
the regulatory mechanisms of SCFAs are extremely complex [13]
[30]
. Future studies will focus on the interaction among SCFAs-associated molecular patterns
and innate immunity in order to clarify the molecular mechanisms behind kidney injury
in the pathogenesis of DN.
